U.S. patent application number 10/741823 was filed with the patent office on 2004-10-07 for ligands of adenine nucleotide translocase (ant) and compositions and methods related thereto.
This patent application is currently assigned to MitoKor, Inc.. Invention is credited to Ghosh, Soumitra S., Pei, Yazhong, Tang, Xiao-Qing.
Application Number | 20040198777 10/741823 |
Document ID | / |
Family ID | 32682231 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040198777 |
Kind Code |
A1 |
Ghosh, Soumitra S. ; et
al. |
October 7, 2004 |
Ligands of adenine nucleotide translocase (ANT) and compositions
and methods related thereto
Abstract
Compounds which have utility in the treatment of conditions
associated with altered mitochondrial function. The compounds have
the following structure (I): 1 including stereoisomers, prodrugs,
and pharmaceutically acceptable salts thereof, wherein X, R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are as defined
herein. Pharmaceutical compositions containing a compound of
structure (I), as well as methods relating to the use thereof, are
also disclosed.
Inventors: |
Ghosh, Soumitra S.; (San
Diego, CA) ; Pei, Yazhong; (San Diego, CA) ;
Tang, Xiao-Qing; (San Diego, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
MitoKor, Inc.
San Diego
CA
|
Family ID: |
32682231 |
Appl. No.: |
10/741823 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60435394 |
Dec 20, 2002 |
|
|
|
Current U.S.
Class: |
514/357 ;
514/408; 514/522; 514/562; 514/563; 514/567; 546/335; 548/577;
558/413; 558/414; 562/430; 562/450 |
Current CPC
Class: |
C07C 311/08 20130101;
C07C 251/24 20130101; C07C 275/42 20130101; C07C 335/16 20130101;
C07C 233/81 20130101; C07C 229/38 20130101 |
Class at
Publication: |
514/357 ;
514/408; 514/522; 514/562; 514/563; 514/567; 546/335; 548/577;
558/413; 558/414; 562/430; 562/450 |
International
Class: |
C07D 213/55; A61K
031/44; A61K 031/40; A61K 031/277; A61K 031/195 |
Claims
What is claimed is:
1. A compound having the following structure: 37or a stereoisomer,
prodrug or pharmaceutically acceptable salt thereof, wherein: X is
--CH.sub.2--Y--, --NH--C(.dbd.Z)--NH--, --CH.dbd.N-- or
--NH--C(.dbd.O)--; Y is --NH--, --S-- or --N(SO.sub.2R.sub.7)--; Z
is O or S; R.sub.1 is hydrogen, halogen, nitro, cyano, alkyl,
substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl,
--NHC(.dbd.O)R', heteroaryl or substituted heteroaryl; R.sub.2,
R.sub.3, R.sub.5 and R.sub.6 are the same or different and
independently hydrogen, halogen, nitro, cyano, alkyl, substituted
alkyl, alkoxy, hydroxy, aryl, substituted aryl, heteroaryl or
substituted heteroaryl; R.sub.4 is hydrogen, halogen, nitro, cyano,
alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,
substituted arylalkyl, heteroaryl, substituted heteroaryl,
heteroarylalkyl, substituted heteroarylalkyl, --O--R.sub.7,
--C(.dbd.O)--R.sub.7, --C(.dbd.O)O--R.sub.7,
--C(.dbd.O)--NH--R.sub.7 or --NHC(.dbd.O)R"; R.sub.7 is hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl or
substituted arylalkyl; R' and R" are the same or different and
independently alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl or substituted heteroaryl; and R.sub.4 and R.sub.5 or
R.sub.5 and R.sub.6, taken together with the carbon atoms to which
they are attached, optionally form a substituted or unsubstituted
homocycle.
2. The compound of claim 1 wherein X is --CH.sub.2--Y-- and Y is
--NH--.
3. The compound of claim 1 wherein X is --CH.sub.2--Y-- and Y is
--S--.
4. The compound of claim 1 wherein X is --CH.sub.2--Y-- and Y is
--N(SO.sub.2R.sub.7)--.
5. The compound of claim 1 wherein X is --NH--C(.dbd.Z)--NH-- and Z
is O.
6. The compound of claim 1 wherein X is --NH--C(.dbd.Z)--NH-- and Z
is S.
7. The compound of claim 1 wherein X is --CH.dbd.N--.
8. The compound of claim 1 wherein X is --NH--C(.dbd.O)--.
9. A composition comprising a compound of claim 1 in combination
with a pharmaceutically acceptable carrier.
10. A method for treating or preventing a condition in a mammal
associated with altered mitochondrial function, comprising
administering to a mammal in need thereof an effective amount of a
composition of claim 9.
11. The method of claim 10 wherein the condition is: Alzheimer's
Disease; diabetes mellitus; obesity; Parkinson's Disease;
Huntington's disease; dystonia; Leber's hereditary optic
neuropathy; schizophrenia; mitochondrial encephalopathy, lactic
acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative
disorders; mitochondrial diabetes and deafness (MIDD); myoclonic
epilepsy ragged red fiber syndrome; osteoarthritis; or Friedrich's
ataxia.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/435,394 filed Dec. 20, 2002, where this
provisional application is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to novel classes of
compounds which interact with adenine nucleotide translocase (ANT),
as well as to compositions and methods for using such compounds to
treat conditions associated with altered mitochondrial
function.
[0004] 2. Description of the Related Art
[0005] Mitochondria are the main energy source in cells of higher
organisms, and these organelles provide direct and indirect
biochemical regulation of a wide array of cellular respiratory,
oxidative and metabolic processes. These include electron transport
chain (ETC) activity, which drives oxidative phosphorylation to
produce metabolic energy in the form of adenosine triphosphate
(ATP), and which also underlies a central mitochondrial role in
intracellular calcium homeostasis.
[0006] Mitochondrial ultrastructural characterization reveals the
presence of an outer mitochondrial membrane that serves as an
interface between the organelle and the cytosol, a highly folded
inner mitochondrial membrane that appears to form attachments to
the outer membrane at multiple sites, and an intermembrane space
between the two mitochondrial membranes. The subcompartment within
the inner mitochondrial membrane is commonly referred to as the
mitochondrial matrix. (For a review, see, e.g., Ernster et al.,
1981 J. Cell Biol. 91:227s). The cristae, originally postulated to
occur as infoldings of the inner mitochondrial membrane, have
recently been characterized using three-dimensional electron
tomography as also including tube-like conduits that may form
networks, and that can be connected to the inner membrane by open,
circular (30 nm diameter) junctions (Perkins et al., 1997, Journal
of Structural Biology 119:260). While the outer membrane is freely
permeable to ionic and non-ionic solutes having molecular weights
less than about ten kilodaltons, the inner mitochondrial membrane
exhibits selective and regulated permeability for many small
molecules, including certain cations, and is impermeable to large
(>.about.10 kDa) molecules.
[0007] Altered or defective mitochondrial activity, including but
not limited to failure at any step of the ETC, may result in the
generation of highly reactive free radicals that have the potential
of damaging cells and tissues. These free radicals may include
reactive oxygen species (ROS) such as superoxide, peroxynitrite and
hydroxyl radicals, and potentially other reactive species that may
be toxic to cells. For example, oxygen free radical induced lipid
peroxidation is a well established pathogenetic mechanism in
central nervous system (CNS) injury such as that found in a number
of degenerative diseases, and in ischemia (i.e., stroke).
[0008] In addition to free radical mediated tissue damage, there
are at least two deleterious consequences of exposure to reactive
free radicals arising from mitochondrial dysfunction that adversely
impact the mitochondria themselves. First, free radical mediated
damage may inactivate one or more of the myriad proteins of the
ETC. Second, free radical mediated damage may result in
catastrophic mitochondrial collapse that has been termed
"permeability transition" (PT) or "mitochondrial permeability
transition" (MPT). According to generally accepted theories of
mitochondrial function, proper ETC respiratory activity requires
maintenance of an electrochemical potential in the inner
mitochondrial membrane by a coupled chemiosmotic mechanism, as
described herein. Free radical oxidative activity, may dissipate
this membrane potential, thereby preventing ATP biosynthesis and
halting the production of a vital biochemical energy source. In
addition, mitochondrial proteins such as cytochrome c may leak out
of the mitochondria after MPT and may induce the genetically
programmed cell suicide sequence known as apoptosis (Wilson, 1998
Cell Death Differen. 5:646-652) or programmed cell death (PCD).
[0009] Altered mitochondrial function characteristic of
mitochondria associated diseases may also be related to loss of
mitochondrial membrane electrochemical potential by mechanisms
other than free radical oxidation, and MPT may result from direct
calcium overload due to excitotoxic mechanisms or indirect effects
of mitochondrial genes, gene products or related downstream
mediator molecules and/or extramitochondrial genes, gene products
or related downstream mediators, or from other known or unknown
causes. Loss of mitochondrial electrochemical potential therefore
may be a critical event in the progression of diseases associated
with altered mitochondrial function, including degenerative
diseases.
[0010] Mitochondrial defects, which may include defects related to
the discrete mitochondrial genome that resides in mitochondrial DNA
and/or to the extramitochondrial genome, which includes nuclear
chromosomal DNA and other extramitochondrial DNA, may contribute
significantly to the pathogenesis of diseases associated with
altered mitochondrial function. For example, alterations in the
structural and/or functional properties of mitochondrial components
comprised of subunits encoded directly or indirectly by
mitochondrial and/or extramitochondrial DNA, including alterations
deriving from genetic and/or environmental factors or alterations
derived from cellular compensatory mechanisms, may play a role in
the pathogenesis of any disease associated with altered
mitochondrial function. A number of diseases and conditions are
thought to be caused by, or to be associated with, alterations in
mitochondrial function. These include: Alzheimer's Disease (AD);
diabetes mellitus; obesity; Parkinson's Disease; Huntington's
disease; dystonia; Leber's hereditary optic neuropathy;
schizophrenia; mitochondrial encephalopathy, lactic acidosis, and
stroke (MELAS); cancer; psoriasis; hyperproliferative disorders;
mitochondrial diabetes and deafness (MIDD); myoclonic epilepsy
ragged red fiber syndrome; osteoarthritis; and Friedrich's ataxia.
The extensive list of additional diseases associated with altered
mitochondrial function continues to expand as aberrant
mitochondrial or mitonuclear activities are implicated in
particular disease processes.
[0011] A hallmark pathology of AD and potentially other diseases
associated with altered mitochondrial function is the death of
selected cellular populations in particular affected tissues.
Mitochondrial dysfunction is thought to be critical in the cascade
of events leading to apoptosis (also referred to as "programmed
cell death" or PCD) in various cell types (Kroemer et al., FASEB J.
9:1277-87, 1995), and may be a cause of apoptotic cell death in
neurons of the AD brain. Altered mitochondrial physiology may be
among the earliest events in PCD (Fiskum et al., J. Cerebral Blood
Flow and Met. 19:351-369, 1999; Murphy et al., J. Cerebral Blood
Flow and Met. 19:231-245, 1999; Zamzami et al., J. Exp. Med.
182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995)
and elevated ROS levels that result from such altered mitochondrial
function may initiate the apoptotic cascade (Ausserer et al., Mol.
Cell. Biol. 14:5032-42,1994).
[0012] Oxidatively stressed mitochondria may release a pre-formed
soluble factor that can induce chromosomal condensation, an event
preceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38,
1996). In addition, members of the Bcl-2 family of anti-apoptosis
gene products are located within the outer mitochondrial membrane
(Monaghan et al., J. Histochem. Cytochem. 40:1819-25, 1992) and
these proteins appear to protect membranes from oxidative stress
(Korsmeyer et al., Biochim. Biophys. Act. 1271:63, 1995).
Localization of Bcl-2 to this membrane appears to be indispensable
for modulation of apoptosis (Nguyen et al., J. Biol. Chem. 269:
16521-24, 1994). Thus, change in mitochondrial physiology may be
important mediators of apoptosis.
[0013] Thus, in addition to their role in energy production in
growing cells, mitochondria (or, at least, mitochondrial
components) participate in cell death (e.g., necrosis and
apoptosis) (Newmeyer et al., 1994, Cell 79:353-364; Liu et al.,
1996, Cell 86:147-157). Apoptosis is apparently also required for,
inter alia, normal development of the nervous system and proper
functioning of the immune system. Moreover, some disease states are
thought to be associated with either insufficient (e.g., cancer,
autoimmune diseases) or excessive (e.g., stroke damage,
AD-associated neurodegeneration) levels of apoptosis. For general
reviews of apoptosis, and the role of mitochondria therein, see
Green and Reed (1998, Science 281:1309-1312), Green (1998, Cell
94:695-698) and Kromer (1997, Nature Medicine 3:614-620). Hence,
agents that effect apoptotic events, including those associated
with mitochondrial components, might have a variety of palliative,
prophylactic and therapeutic uses.
[0014] The adenine nucleotide translocase (ANT), a nuclear encoded
mitochondrial protein, is reportedly the most abundant protein of
the inner mitochondrial membrane, forming dimers that comprise up
to 10% of the total mitochondrial protein in highly oxidative
tissue like cardiac and skeletal muscle. Three human ANT isoforms
have been described that appear to differ in their tissue
expression patterns, and other mammalian ANT homologues have been
described. See, e.g., Wallace et al., 1998 in Mitochondria &
Free Radicals in Neurodegenerative Diseases, Beal, Howell and
Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 283-307, and
references cited therein. ANT proteins mediate the exchange across
the mitochondrial inner membrane of ATP synthesized in the
mitochondrial matrix for adenosine diphosphate (ADP) in the
cytosol. This nucleotide exchange is the most active transport
system in aerobic cells, and is a critical component in maintaining
cellular energy metabolism (for a review see Klingenberg, J.
Bioenergetics and Biomembranes 25:447-457, 1993). ANT has also been
implicated as an important molecular component of the MPT pore, a
Ca.sup.2+-regulated inner membrane channel that plays an important
modulating role in apoptotic processes.
[0015] By way of background, all five of the multisubunit complexes
that mediate ETC activity are localized to the inner mitochondrial
membrane. ANT represents the most abundant of the inner
mitochondrial membrane proteins. In at least three distinct
chemical reactions known to take place within the ETC,
positively-charged protons are moved from the mitochondrial matrix,
across the inner membrane, to the intermembrane space. This
disequilibria of charged species creates an electrochemical
potential of approximately 220 mV referred to as the "protonmotive
force" (PMF), which is often represented by the notation
.DELTA..psi. and represents the sum of the electric potential and
the pH differential across the inner mitochondrial membrane (see,
e.g., Ernster et al., 1981 J. Cell Biol. 91:227s and references
cited therein).
[0016] This membrane potential drives the ANT-mediated
stoichiometric exchange of ATP and ADP across the inner
mitochondrial membrane, and provides the energy contributed to the
phosphate bond created when ADP is phosphorylated to yield ATP by
ETC Complex V, a process that is "coupled" stoichiometrically with
transport of a proton into the matrix. Under normal metabolic
conditions, the inner membrane is impermeable to proton movement
from the intermembrane space into the matrix, leaving ETC Complex V
as the sole means whereby protons can return to the matrix. When,
however, the integrity of the inner mitochondrial membrane is
compromised, as occurs during MPT, which may accompany a disease
associated with altered mitochondrial function, protons are able to
bypass the conduit of Complex V without generating ATP, thereby
"uncoupling" respiration because electron transfer and associated
proton pumping yields no ATP. Thus, MPT involves the opening of a
mitochondrial membrane "pore", a process by which, inter alia, the
ETC and ATP synthesis are uncoupled, .DELTA..psi.m collapses and
mitochondrial membranes lose the ability to selectively regulate
permeability to solutes both small (e.g., ionic Ca.sup.2+,
Na.sup.+, K.sup.+, H.sup.+) and large (e.g., proteins)
molecules.
[0017] Without wishing to be bound by theory, it is unresolved
whether this pore is a physically discrete conduit that is formed
in mitochondrial membranes, for example by assembly or aggregation
of particular mitochondrial and/or cytosolic proteins and possibly
other molecular species, or whether the opening of the "pore" may
simply represent a general increase in the porosity of the
mitochondrial membrane.
[0018] MPT may also be induced or blocked by compounds that bind
one or more mitochondrial molecular components. Such compounds
include, but are not limited to, atractyloside and bongkrekic acid,
which are known to bind to ANT. Methods of determining appropriate
amounts of such compounds to induce MPT are known in the art (see,
e.g., Beutner et al., 1998 Biochim. Biophys. Acta 1368:7; Obatomi
and Bach, 1996 Toxicol. Lett. 89:155; Green and Reed, 1998 Science
281:1309; Kroemer et al., 1998 Annu. Rev. Physiol. 60:619; and
references cited therein). Thus, certain mitochondrial molecular
components, such as ANT, may contribute to the MPT mechanism.
[0019] It is known that when fatty acids bind to ANT, they can
induce what is termed "mild" mitochondrial uncoupling. In
bioenergetic terms, the word "mild" means that the uncoupling is
only evident at the resting state of the mitochondria (i.e. state
4, nonphosphorylating respiration) when the membrane potential is
maximal, and that there is little or no effect during robust ATP
production. Additionally, it has been discovered that this
uncoupling induced by free fatty acids may be reversed by the
addition of the ANT ligand carboxyatractyloside (see e.g., Andreyev
et al., 1989 Eur. J. Biochem. 182:585-592; Skulachev, 1991 FEBS
Lett. 294:158-162; Skulachev, 1996 FEBS Leff. 397:7-10; Korshunov
et al., 1998 FEBS Lett. 435:215-218; Wojtczak et al., 1998 Archives
of Biochem. and BioPhys. 357:76-84; and references cited therein),
suggesting that carboxyatractyloside blocks the proton conductance
induced by the free fatty acid. Since the high membrane potential
in the resting state of the mitochondria potentiates mitochondrial
free radical production (see e.g., Boveris and Chance, 1973
Biochem. J. 134:707-716; Korshunov et al., 1997 FEBS Lett.
416:15-18; and references cited therein), it has been theorized
that periods of mild uncoupling may serve the purpose of reducing
oxidative stress and could slow the rate of Ca.sup.2+ uptake at
high membrane potential (Skulachev, 1996 FEBS Lett. 397:7-10;
Korshunov et al., 1997 FEBS Lett. 416:15-18; and references cited
therein).
[0020] ANT proteins, as well as other transporter proteins known
more generally as uncoupling proteins (UCPs), belong to a larger
family of proteins known as the "carrier" family. The theory that
mild uncoupling may be induced via brain-specific isoforms of UCPs
has recently become the focus of several studies (Yu et al., 2000
FASEB J. 14(11):1611-1618; Farrelly et al., 2001 Analytical
Biochem. 293:269-276; and references cited therein). Additionally,
mild mitochondrial uncoupling has recently been proposed as a
possible treatment for ischemia-reperfusion injury (Morin et al.,
2001 Advanced Drug Delivery Rev. 49:151-174; and references cited
therein).
[0021] Clearly there is a need for compounds and methods that limit
or prevent damage to organelles, cells and tissues that may
directly or indirectly result from alterations in mitochondrial
function including mitochondrial dysfunction, such as mitochondrial
permeability transition that is the cause or consequence of
oxidative phosphorylation uncoupling and/or intracellular free
radical generation. Accordingly, while significant advances have
been made in this field, there is still a need in the art for small
molecules that will bind, form a complex with, or otherwise
interact with ANT. There is also a need for pharmaceutical
compositions containing the same, as well as methods relating to
the use thereof to treat conditions associated with altered
mitochondrial function. The present invention fulfills these needs
and provides other related advantages.
BRIEF SUMMARY OF THE INVENTION
[0022] This invention is directed to novel classes of compounds
which bind, form a complex with, or otherwise interact with ANT.
This invention is also directed to compositions containing one or
more of such compounds in combination with one or more
pharmaceutically acceptable carriers, as well as to methods for
treating or preventing conditions associated with altered
mitochondrial function with such compounds. Without wishing to be
bound by theory, it is believed that the compounds of the present
invention interact with ANT to induce mild mitochondrial
uncoupling.
[0023] In one embodiment, this invention is directed to compounds
which have the following structure (I): 2
[0024] including stereoisomers, prodrugs, and pharmaceutically
acceptable salts thereof, wherein X, R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 are as defined herein.
[0025] In addition, compositions containing a compound of this
invention in combination with a pharmaceutically acceptable carrier
are disclosed. Methods of use for treating or preventing conditions
associated with altered mitochondrial function, the treatment or
prevention of which may be effected or facilitated by inducing mild
mitochondrial uncoupling, with the compounds of this invention and
compositions comprising them are also disclosed. In particular,
methods of use for the treatment and prevention of Alzheimer's
Disease, diabetes mellitus, obesity, Parkinson's Disease,
Huntington's disease, dystonia, Leber's hereditary optic
neuropathy, schizophrenia, mitochondrial encephalopathy, lactic
acidosis, and stroke (MELAS), cancer, psoriasis, hyperproliferative
disorders, mitochondrial diabetes and deafness (MIDD), myoclonic
epilepsy ragged red fiber syndrome, osteoarthritis and Friedrich's
ataxia, as well as other conditions associated with altered
mitochondrial function with the compounds of this invention and
compositions comprising them are disclosed.
[0026] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, various references are set forth
herein which describe in more detail certain aspects of this
invention, and are incorporated by reference in their
entireties
DETAILED DESCRIPTION OF THE INVENTION
[0027] In one embodiment of the present invention, compounds are
disclosed having the following structure (I): 3
[0028] or a stereoisomer, prodrug or pharmaceutically acceptable
salt thereof,
[0029] wherein:
[0030] X is --CH.sub.2--Y--, --NH--C(.dbd.Z)--NH--, --CH.dbd.N-- or
--NH--C(.dbd.O)--;
[0031] Y is --NH--, --S-- or --N(SO.sub.2R.sub.7)--;
[0032] Z is O or S;
[0033] R.sub.1 is hydrogen, halogen, nitro, cyano, alkyl,
substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl,
--NHC(.dbd.O)R', heteroaryl or substituted heteroaryl;
[0034] R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are the same or
different and independently hydrogen, halogen, nitro, cyano, alkyl,
substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl,
heteroaryl or substituted heteroaryl;
[0035] R.sub.4 is hydrogen, halogen, nitro, cyano, alkyl,
substituted alkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,
substituted heteroarylalkyl, --O--R.sub.7, --C(.dbd.O)--R.sub.7,
--C(.dbd.O)O--R.sub.7, --C(.dbd.O)--NH--R.sub.7 or
--NHC(.dbd.O)R";
[0036] R.sub.7 is hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, arylalkyl or substituted arylalkyl;
[0037] R' and R" are the same or different and independently alkyl,
substituted alkyl, aryl, substituted aryl, heteroaryl or
substituted heteroaryl; and
[0038] R.sub.4 and R.sub.5 or R.sub.5 and R.sub.6, taken together
with the carbon atoms to which they are attached, optionally form a
substituted or unsubstituted homocycle.
[0039] As used herein, the above terms have the meanings set forth
below.
[0040] "Alkyl" means a straight chain or branched, noncyclic or
cyclic, unsaturated or saturated aliphatic hydrocarbon containing
from 1 to 10 carbon atoms. Representative saturated straight chain
alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,
and the like; while saturated branched alkyls include isopropyl,
sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Unsaturated alkyls contain at least one double or triple bond
between adjacent carbon atoms (referred to as an "alkenyl" or
"alkynyl", respectively). Representative straight chain and
branched alkenyls include ethylenyl, propylenyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like; while representative straight chain and branched alkynyls
include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,
2-pentynyl, 3-methyl-1 butynyl, and the like. Representative
saturated cyclic alkyls include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, --CH.sub.2cyclohexyl, and the like; while
unsaturated cyclic alkyls include cyclopentenyl, cyclohexenyl,
--CH.sub.2cyclohexenyl, and the like.
[0041] "Aryl" means an aromatic carbocyclic moiety such as phenyl
or naphthyl (i.e., 1- or 2-naphthyl).
[0042] "Arylalkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with an aryl moiety, such as benzyl,
--(CH.sub.2).sub.2phenyl, --(CH.sub.2).sub.3phenyl, and the
like.
[0043] "Heteroaryl" means an aromatic heterocycle ring of 5 to 10
members and having at least one heteroatom selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and bi-cyclic ring systems. Representative heteroaryls
are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,
quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl,
benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl.
[0044] "Heteroarylalkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heteroaryl moiety, such as
--(CH.sub.2).sub.2pyridyl- , --(CH.sub.2).sub.3pyridyl, and the
like.
[0045] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 to 4
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Heterocycles include
heteroaryls as defined above. Thus, in addition to the heteroaryls
listed above, heterocycles also include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl,
valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0046] "Homocycle" means a saturated, unsaturated or aromatic
carbocyclic moiety.
[0047] The term "substituted" as used herein means any of the above
groups (i.e., alkyl, aryl, arylalkyl, heteroaryl heteroarylalkyl,
heterocycle and homocycle) wherein at least one hydrogen atom is
replaced with a substituent. In the case of an oxo substituent
(".dbd.O"), two hydrogen atoms are replaced. When substituted,
"substituents" within the context of this invention include oxo,
halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino,
alkyl, substituted alkyl, alkoxy, thioalkyl, sulfonylalkyl,
haloalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,
substituted heteroarylalkyl, heterocycle, substituted heterocycle,
homocycle, substituted homocycle, --NR.sub.aR.sub.b,
--NR.sub.aC(.dbd.O)R.sub.b, --NR.sub.aC(.dbd.O)NR.sub.aNR.sub.b,
--NR.sub.aC(.dbd.O)OR.sub.b, --NR.sub.aSO.sub.2R.sub.b,
--C(.dbd.O)R.sub.a, --C(.dbd.O)OR.sub.a,
--C(.dbd.O)NR.sub.aR.sub.b, --OC(.dbd.O)NR.sub.aR.sub.b,
--OR.sub.a, --SR.sub.a, --SOR.sub.a, --S(.dbd.O).sub.2R.sub.a,
--OS(.dbd.O).sub.2R.sub.a, --S(.dbd.O).sub.2OR.sub.a,
--CH.sub.2S(.dbd.O).sub.2R.sub.a,
--CH.sub.2S(.dbd.O).sub.2N(R.sub.a).sub.2,
.dbd.NS(.dbd.O).sub.2R.sub.a, and
--S(.dbd.O).sub.2N(R.sub.a).sub.2, wherein R.sub.a and R.sub.b are
the same or different and independently hydrogen, alkyl,
substituted alkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,
substituted heteroarylalkyl, heterocycle, substituted heterocycle,
homocycle or substituted homocycle.
[0048] "Alkoxy" means an alkyl moiety attached through an oxygen
bridge (i.e., --O-alkyl) such as methoxy, ethoxy, and the like.
[0049] "Alkylamino" and "dialkylamino" mean one or two alkyl
moieties attached through a nitrogen bridge (i.e., --N-alkyl) such
as methylamino, ethylamino, dimethylamino, diethylamino, and the
like.
[0050] "Haloalkyl" means an alkyl moiety having at least one alkyl
hydrogen replaced with halogen, such as --CF.sub.3, and the
like.
[0051] "Halogen" means fluoro, chloro, bromo and iodo.
[0052] "Sulfonylalkyl" means an alkyl moiety attached through a
sulfonyl bridge (i.e., --SO.sub.2-alkyl) such as methylsulfonyl,
ethylsulfonyl, and the like.
[0053] "Thioalkyl" means an alkyl moiety attached through a sulfur
bridge (i.e., --S-alkyl) such as methylthio, ethylthio, and the
like.
[0054] In a further embodiment of structure (I), X is
--CH.sub.2--Y--, Y is --NH-- and compounds of this invention have
the following structure (II): 4
[0055] In another further embodiment of structure (I), X is
--CH.sub.2--Y--, Y is --S-- and compounds of this invention have
the following structure (III): 5
[0056] In still another further embodiment of structure (I), X is
--CH.sub.2--Y--, Y is --N(SO.sub.2R.sub.7)-- and compounds of this
invention have the following structure (IV): 6
[0057] In still another further embodiment of structure (I), X is
--NH--C(.dbd.Z)--NH--, Z is O and compounds of this invention have
the following structure (V): 7
[0058] In still another further embodiment of structure (I), X is
--NH--C(.dbd.Z)--NH--, Z is S and compounds of this invention have
the following structure (VI): 8
[0059] In still another further embodiment of structure (I), X is
--CH.dbd.N-- and compounds of this invention have the following
structure (VII): 9
[0060] In still another further embodiment of structure (I), X is
--NH--C(.dbd.O)-- and compounds of this invention have the
following structure (VIII): 10
[0061] The compounds of the present invention may generally be
utilized as the free acid or free base. Alternatively, the
compounds of this invention may be used in the form of acid or base
addition salts. Acid addition salts of the free amino compounds of
the present invention may be prepared by methods well known in the
art, and may be formed from organic and inorganic acids. Suitable
organic acids include maleic, fumaric, benzoic, ascorbic, succinic,
methanesulfonic, acetic, oxalic, propionic, tartaric, salicylic,
citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic,
palmitic, glycolic, glutamic, and benzenesulfonic acids. Suitable
inorganic acids include hydrochloric, hydrobromic, sulfuric,
phosphoric, and nitric acids. Base addition salts include those
salts that form with the carboxylate anion and include salts formed
with organic and inorganic cations such as those chosen from the
alkali and alkaline earth metals (for example, lithium, sodium,
potassium, magnesium, barium and calcium), as well as the ammonium
ion and substituted derivatives thereof (for example,
dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, and the
like). Thus, the term "pharmaceutically acceptable salt" of
structure (I) is intended to encompass any and all acceptable salt
forms.
[0062] In addition, prodrugs are also included within the context
of this invention. Prodrugs are any covalently bonded carriers that
release a compound of structure (I) in vivo when such prodrug is
administered to a patient. Prodrugs are generally prepared by
modifying functional groups in a way such that the modification is
cleaved, either by routine manipulation or in vivo, yielding the
patent compound. Prodrugs include, for example, compounds of this
invention wherein hydroxy or amine groups are bonded to any group
that, when administered to a patient, cleaves to form the hydroxy
or amine groups. Thus, representative examples of prodrugs include
(but are not limited to) acetate, formate and benzoate derivatives
of alcohol and amine functional groups of the compounds of
structure (I). Further, in the case of a carboxylic acid (--COOH),
esters may be employed, such as methyl esters, ethyl esters, and
the like.
[0063] With regard to stereoisomers, the compounds of structure (I)
may have chiral centers and may occur as racemates, racemic
mixtures and as individual enantiomers or diastereomers. All such
isomeric forms are included within the present invention, including
mixtures thereof. Furthermore, some of the crystalline forms of the
compounds of structure (I) may exist as polymorphs, which are
included in the present invention. In addition, some of the
compounds of structure (I) may also form solvates with water or
other organic solvents. Such solvates are similarly included within
the scope of this invention.
[0064] The compounds of the present invention may be prepared by
known organic synthesis techniques, including the methods described
in more detail in the Examples.
[0065] Activities of the compounds of the present invention are
typically calculated from the IC.sub.50 as the concentration of a
compound necessary to displace 50% of a detectable (i.e.,
detectably labeled, for example, radiolabeled) ligand from ANT
molecules, which may be present as isolated or purified
polypeptides or as components of preparations containing isolated
mitochondria or submitochondrial particles (SMP) using established
ligand binding assays or modifications thereof. For example,
compounds may be tested for their ability to compete with
radiolabeled atractyloside (ATR), or with a radiolabeled ATR
derivative, for binding to isolated ANT polypeptides or to ANT
present in isolated mitochondria or SMP.
[0066] As another example, the relative affinities for ANT of
various compounds of the present invention may be determined by a
fluorescence assay that exploits the fluorescent properties of an
ATR derivative. When such an ATR derivative is bound to ANT, the
fluorescence is quenched. When, however, such an ATR derivative is
displaced from ANT by a known concentration of ATR or an ATR
derivative that is an ANT ligand, fluorescence dequenching that
results from displacement of the fluorophore can be measured in
real time.
[0067] Briefly, a mitochondrial preparation is washed and
resuspended in a suitable buffer in the presence of an ATR
derivative with fluorescent properties, washed to remove unbound
fluorophore and placed in a fluorometer equipped with a light
source and filter set appropriate for the fluorophore. Fluorescence
intensity is monitored as a function of time, and a candidate
compound is then added to determine its ability to compete with the
ATR derivative for binding to ANT, as evidenced by a change in
detectable relative fluorescence intensity units. After the
fluorescence signal has stabilized, any additional ATR derivative
that remains bound to ANT is displaced by adding an excess (e.g.,
.mu.M quantities) of ATR as a competitive inhibitor, to determine
maximal signal intensity and therefrom calculate the proportion of
the ATR derivative displaced by the candidate compound. Those
having familiarity with the art will appreciate that variations and
modifications may be made to ANT-binding assays such as those
illustrated above and described in the Examples for determining the
activities and IC.sub.50 values of candidate compounds, and which
are not intended to be limiting. See also U.S. Ser. No. 09/569,327
entitled "Production of Adenine Nucleotide Translocator (ANT),
Novel ANT Ligands and Screening Assays Therefor", which is hereby
incorporated by reference.
[0068] As mentioned above, it is believed that the compounds of
this invention bind, form a complex with, or otherwise interact
with ANT to induce mild mitochondrial uncoupling, and are thereby
useful in the treatment of a variety of conditions associated with
altered mitochondrial function. In this regard, the compounds of
this invention have utility over a broad range of therapeutic
applications, and may be used to treat conditions including (but
not limited to) Alzheimer's Disease, diabetes mellitus, obesity,
Parkinson's Disease, Huntington's disease, dystonia, Leber's
hereditary optic neuropathy, schizophrenia, mitochondrial
encephalopathy, lactic acidosis, and stroke (MELAS), cancer,
psoriasis, hyperproliferative disorders, mitochondrial diabetes and
deafness (MiDD), myoclonic epilepsy ragged red fiber syndrome,
osteoarthritis and Friedrich's ataxia.
[0069] The compounds of the present invention are preferably part
of a pharmaceutical composition when used in the methods of the
present invention. The pharmaceutical composition will include at
least one of a pharmaceutically acceptable carrier, dilutent or
excipient, in addition to a therapeutically effective amount of one
or more compounds of the present invention and, optionally, other
components.
[0070] The "therapeutically effective amount" of a compound of the
present invention will depend on the route of administration, the
type of warm-blooded mammal being treated, and the physical
characteristics of the specific mammal under consideration. These
factors and their relationship to determining this amount are well
known to skilled practitioners in the medical arts. Furthermore.,
this amount and the method of administration can be tailored to
achieve optimal efficacy but will depend on such factors as weight,
diet, concurrent medication and other factors which as noted those
skilled in the medical arts will recognize. Typically, dosages will
be between about 0.01 mg/kg and 100 mg/kg body weight, preferably
between about 0.01 and 10 mg/kg body weight.
[0071] "Pharmaceutically acceptable carriers" for therapeutic use
are well known in the pharmaceutical art. For compositions
formulated as liquid solutions, acceptable carriers and/or diluents
include saline and sterile water, and may optionally include
antioxidants, buffers, bacteriostats and other common additives.
Preservatives, stabilizers, dyes and even flavoring agents may also
be provided in the pharmaceutical composition. The compositions can
also be formulated as pills, capsules, granules, or tablets that
contain, in addition to a compound of this invention, dispersing
and surface active agents, binders, and lubricants. One skilled in
this art may further formulate a compound in an appropriate manner,
and in accordance with accepted practices, such as those disclosed
in Remington's Pharmaceutical Sciences, Gennera, Ed., Mack
Publishing Co., Easton, Pa. 1990.
[0072] The pharmaceutical compositions that contain one or more
compounds that interact with ANT may be in any form which allows
for the composition to be administered to a patient. For example,
the composition may be in the form of a solid, liquid or gas
(aerosol). Typical routes of administration include, without
limitation, oral, topical, parenteral (e.g., sublingually or
buccally), sublingual, rectal, vaginal, and intranasal. The term
parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal, intracavernous,
intrameatal, intraurethral injection or infusion techniques. The
pharmaceutical composition is formulated so as to allow the active
ingredients contained therein to be bioavailable upon
administration of the composition to a patient. Compositions that
will be administered to a patient take the form of one or more
dosage units, where for example, a tablet may be a single dosage
unit, and a container of one or more compounds of the invention in
aerosol form may hold a plurality of dosage units.
EXAMPLES
[0073] The following Examples are offered for purposes of
illustration, not limitation. Those skilled in the art will
recognize, or be able to ascertain through routine experimentation,
numerous equivalents to the specific substances and procedures
described herein. Such equivalents are considered to be within the
scope of the present invention.
1TABLE 1 Abbreviations used in Examples Reagents: DIEA
diisopropylethylamine DMAP 4-N,N,-dimethylaminopyridine TFA
trifluoroacetic acid Solvents: DCM dichloromethane DMF
dimethylformamide DMSO dimethylsulfoxide EtOAc ethyl acetate MeOH
methanol THF tetrahydrofuran Others: rt room temperature g gram hr
hour min minute
Example 1
Synthesis of Representative Compounds
[0074] These examples illustrate the preparation of certain
representative compounds.
[0075] A. General Synthesis of 3-Substituted-Amido, -Ureido and
-Thioureido Analogs of Salicylic Acid 11
[0076] To a solution of 3-aminosalicylic acid 1 (0.05 g, 0.326
mmol) and DIEA (227 .mu.l, 1.63 mmol) in dry dichloromethane (2 ml)
was added p-tolyl isocyanate (82.2 ul, 0.652 mmol) at rt, and the
mixture was stirred for 3 hrs. The mixture was then quenched by
addition of 10% water in methanol and stirred at rt for 1 hr. The
mixture was concentrated under reduced pressure, the residue was
diluted with 0.5 M NaOH and then extracted twice with ether. The
aqueous layer was acidified with concentrated HCI and extracted
with ethyl acetate. The organic layers were combined, washed with
brine and dried over anhydrous sodium sulfate. The solvent was
evaporated in vacuo to provide structure 2 (X=O and
R=4-methylphenyl) in 90% yield. The purity of the product was
checked by LC/MS.
[0077] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 8.26 (d, J=8.1 Hz,
1H), 7.51 (d, J=8.1 Hz, 1H), 7.31 (d, J=6.8 Hz, 2H), 7.10 (d, J=8.2
Hz, 2H), 6.87 (t, J=8.1 Hz, 1H), 2.29 (s, 3H); MS (calculated for
C.sub.15H.sub.15N.sub.2O.sub.4) 287.1 [M+H].sup.+, found 287.0.
[0078] The foregoing procedure is also illustrative of the
synthesis of thiourea and amide analogs. In most cases, products
obtained were very pure and did not require further purification.
However, if necessary, a RP-HPLC using a C8 column (Betasil C18,
150.times.70 mm, (Thermo Hypersil-Keystone, Bellefonte, Pa.);
solvent A: 0.05% TFA in H.sub.2O, solvent B: 0.05% TFA in
CH.sub.3CN; gradient: 5-95% B over 40 min) was used to purify
products to homogeneity.
[0079] B. General Synthesis of 3-Arylaminomethyl and
3-Aryliminomethyl Analogs of Salicylic Acid 12
[0080] Synthesis of Structure 4 (R=H):
[0081] A stirred solution of 3-formyl-2-hydroxybenzoic acid 3 (83
mg, 0.5 mmol) and aniline (49 mg, 0.53 mmol) in toluene (5 mL) was
refluxed overnight. After it was cooled to rt, the red precipitate
was collected by filtration, washed with benzene, and dried under
vacuum to give structure 4 (R=H) (110 mg, 91 %) as a red solid.
.sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 6.88 (t, J=7.4 Hz, 2H),
7.42 (t, J=7.3 Hz, 1H), 7.55 (t, J=7.6 Hz, 2H), 7.65 (d, J=8.1 Hz,
2H), 7.92 (dd, J=1.8 Hz, 7.9 Hz, 1H), 8.10 (dd, J=1.8 Hz, 7.9 Hz,
1H), 9.28 (s, 1H); MS (calculated for C.sub.14H.sub.12NO.sub.3)
242.07 [M+H].sup.+, found 242.0.
[0082] Synthesis of Structure 5 (R=H):
[0083] To a solution of structure 4 (50 mg, 0.21 mmol) in methanol
(5 mL) was added NaBH.sub.3CN (65 mg, 1.0 mmol). The reaction
mixture was stirred at rt for 0.5 hrs and then quenched with
H.sub.2O. The solvent was evaporated under reduced pressure, and
the residue was extracted with EtOAc three times. The combined
organic layer was washed with saturated brine, dried over
Na.sub.2SO.sub.4, filtered, concentrated, and the residue was
crystallized from methanol to give structure 5 (R=H) (32 mg, 63%)
as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 4.25
(s, 2H), 6.51 (t, J=7.3 Hz, 1H), 6.55 (d, J=7.9 Hz, 2H), 6.87 (t,
J=7.7 Hz, 1H), 7.04 (t, J=7.7 Hz, 2H), 7.47 (d, J=7.2 Hz, 1H), 7.69
(d, J=7.7 Hz, 1H); MS (calculated for C.sub.14H.sub.14NO.sub.3)
244.09 [M+H].sup.+, found 243.9.
[0084] C. General Synthesis of 3-Arylsulfanylmethyl Analogs of
5-Methyl Salicylic Acid 13
[0085] Synthesis of 6 (Ruell et al., J. Org. Chem.
64:5858-5866.1999):
[0086] A solution of 5-methylsalicylic acid (5 g, 33 mmol) and
hexamethylenetetramine (22 g, 150 mmol) in TFA was warmed to
90.degree. C. and stirred for 14 hrs. The orange solution that
resulted was poured into dilute hydrochloric acid (1 M, 500 mL) and
the solution was stirred for a further 6 hrs. The white precipitate
was filtered and then dried in a vacuum desiccator for 2 days.
Structure 6 (6.7 g) was obtained as a damp off-white solid.
[0087] Synthesis of Structure 7:
[0088] To a stirred solution of structure 6 (3.0 g, 16.6 mmol)
dissolved in dichloromethane (140 mL) was added
diisopropylethylamine (7.2 mL, 41.6 mmol). The solution was cooled
to 0C and chloromethyl methyl ether (2.8 mL, 36.9 mmol) was added
drop-wise. After 30 min, the reaction mixture was warmed to room
temperature and stirred for an additional 1 hr. A saturated
solution of sodium bicarbonate (50 mL) was added and the mixture
was vigorously stirred for 10 min. The organic phase was removed
and the aqueous phase was extracted with dichloromethane (20 mL).
The combined organic phase was dried over sodium sulfate, filtered
and evaporated to give structure 7 (2.47 g, 55% yield) as a yellow
oil. R.sub.f [40-60 petroleum ether:ethyl acetate (4:1)]=0.48;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.41 (s, 3H), 3.56 (s,
3H), 3.57 (s, 3H), 5.17 (s, 2H), 5.48 (s, 2H), 7.85 (d, J=2.0 Hz,
1H), 7.95 (d, J=2.0 Hz, 1H), 10.41 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 20.5, 58.0, 91.3, 102.4, 124.8, 130.9, 132.6,
134.4, 137.9, 158.5, 164.4, 199.1; LC/MS 269.4 [M+H].sup.+, 286.1
[M+NH.sub.4].sup.+, 554.4[2M+NH.sub.4].sup.+.
[0089] Synthesis of Structure 8:
[0090] To a solution of structure 7 (2.45 g, 9.1 mmol) in methanol
(50 mL) at 0.degree. C. was added sodium borohydride (0.50 g, 13
mmol). The solution was warmed to room temperature and stirring was
continued for 30 min. The reaction mixture was diluted with brine
(250 mL) and extracted with ethyl acetate (3.times.100 mL). The
combined organic phase was dried over sodium sulfate, filtered and
evaporated to give structure 8 (2.32 g, 94% yield) as a colorless
oil. R.sub.f [40-60 petroleum ether:ethyl acetate (4:1)]=0.21;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.28 (s, 3H), 3.31 (brs,
1H), 3.48 (s, 3H), 3.54 (s, 3H), 4.55 (d, J=6.0 Hz, 2H), 5.02 (s,
2H), 5.38 (s, 2H), 7.32 (d, J=2.0 Hz, 1H), 7.61 (d, J=2.0 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 20.6, 57.4, 57.8, 60.8,
91.0, 101.5, 123.4, 131.7, 134.3, 135.6, 136.1, 154.8, 165.0; LC/MS
271.3 [M+H].sup.+, 558.4 [2M +NH.sub.4].sup.+.
[0091] Synthesis of Structure 9:
[0092] To a stirred solution of triphenylphosphine (728 mg, 2.77
mmol) in dichloromethane (5.0 mL) at room temperature was added
imidazole (189 mg, 2.77 mmol) and iodine (704 mg, 2.77 mmol),
followed by a solution of structure 8 (500 mg, 1.85 mmol) dissolved
in dichloromethane (2.0 mL) and stirring was continued for 1 hr.
The reaction mixture was loaded directly onto silica and eluted
with 40-60 petroleum ether:ethyl acetate (6:1) to give structure 9
(450 mg, 64% yield) as a colorless oil. R.sub.f [40-60 petroleum
ether:ethyl acetate (4:1)]=0.60; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 2.32 (s, 3H), 3.55 (s, 3H), 3.63 (s, 3H), 4.56 (s, 2H),
5.15 (s, 2H), 5.44 (s, 2H), 7.38 (d, J=2.0 Hz, 1H), 7.59 (d, J=2.0
Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. -0.2, 20.5,
57.7, 57.9, 91.1, 101.1, 124.6, 131.9, 134.1, 134.2,
135.6,153.6,165.0; LC/MS 381.0 [M+H].sup.+, 398.4
[M+NH.sub.4].sup.+, 778.0 [2M+NH.sub.4].sup.+.
[0093] Synthesis of Structure 10 (R=2-pyrimidinyl: X=S):
[0094] To a vigorously stirred solution of potassium hydride (4.7
mg, 117 .mu.mol) in THF (0.5 mL) under a nitrogen atmosphere at
0.degree. C. was added 2-mercaptopyrimidine (11.4 mg, 102 .mu.mol).
Stirring was continued for 10 min at 0.degree. C. and then
structure 9 (38.8 mg, 102 82 mol) in THF (0.5 mL) was added. The
reaction mixture was placed in a cold room (-10.degree. C.)
overnight (14 hrs). The reaction mixture was then diluted with
brine (2 mL) and extracted with dichloromethane (2.times.3 mL). The
combined organic phase was dried with sodium sulfate, filtered and
evaporated under a stream of nitrogen. The resultant oil was
dissolved in acetonitrile:water:concentrated HCl (90:9:1) and
heated to 65.degree. C. The reaction mixture was then frozen and
lyophilized to give structure 10 (R=2-pyrimidinyl) (32.2 mg, 86%
yield) as an off white powder. .sup.1H NMR (400 MHz,
CDCl.sub.3/CD.sub.3OD) .delta. 2.25 (s, 3H), 4.48 (s, 2H), 7.04 (t,
J=4.7 Hz, 1H), 7.46 (s, 1H), 7.61 (s, 1H), 8.56 (d, J=4.7 Hz, 2H);
ESMS m/z 277.3 [M+H].sup.+; LC/MS 277.2 [M+H].sup.+, 553.3
[2M+H].sup.+.
[0095] D. Synthesis of 3-Arylsulfonamidomethyl Analogs of 5-Alkyl
Salicylic Acid 14
[0096] Synthesis of Structure 11:
[0097] To a stirred solution of structure 6 (3.75 g, 20.8 mmol) in
diethyl ether (2.5 mL) and ethyl acetate (2.5 mL) was added
diazomethane (generated from Diazald (20 g), according to Aldrich
Technical Notes #AL-180) drop-wise. The solvent and excess
diazomethane were evaporated to give structure 11 (4.03 g, 100%
yield) as a pale yellow solid. R.sub.f (40-60 petroleum ether:ethyl
acetate (9:1))=0.30; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.32
(s, 3H), 3.98 (s, 3H), 7.81 (d, J=2 Hz, 1H), 7.90(d, J=2Hz, 1H),
10.49 (s, 1H), 11.3 (s, 1H).
[0098] Synthesis of Structure 12 (R.sup.1=4-tert-butylphenyl):
[0099] To a solution of structure 11 (806 mg, 4.15 mmol) in
methanol (15 mL) at 0.degree. C. was added 4-tert-butylaniline
(1.24 ml, 7.79 mmol) then sodium cyanoborohydride (265 mg, 4.21
mmol). The mixture was warmed to room temperature and stirred for
20 hrs. The reaction mixture was diluted with dichloromethane (10
mL) and an aqueous solution of hydrochloric acid (2 mol/L, 10 mL)
was added. Stirring was continued for a further 30 min then the
reaction mixture was cooled to 0.degree. C. and neutralized by the
drop-wise addition of aqueous sodium hydroxide solution (2 M). The
organic phase was removed and the aqueous phase extracted with
further dichloromethane (10 mL). The combined organic phase was
washed with water (10 mL) and brine (10 mL) then dried with sodium
sulfate, filtered and evaporated. Purification on silica (5 g) with
40-60 petroleum ether:ethyl acetate 10:1, 8:1 then 3:2) provided
structure 12 (R.sup.1=4-tert-butylphenyl) (680 mg, 50%) as a cream
solid. R.sub.f (40-60 petroleum ether:ethyl acetate (9:1))=0.75;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.28 (s, 9H), 2.25 (s,
3H), 3.93 (s, 3H), 4.34 (s, 2H), 6.62 (d, J=8.0 Hz, 2H), 7.19 (d,
J=8.0 Hz, 2H), 7.34 (s, 1H), 7.55 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 21.2, 32.2, 34.5, 44.1, 52.9, 112.4, 113, 6,
126.7, 128.1, 128.6, 129.1, 136.6, 146.5, 158.3, 171.5; ESMS m/z
328.6 [M+H].sup.+, 369.5 [M+CH.sub.3CN+H].sup.+, 655.6
[2M+H].sup.+.
[0100] Synthesis of Structure 13 (R.sup.1=4-tert-butylphenyl,
R.sup.2=CH.sub.3):
[0101] To a stirred solution of structure 12
(R.sup.1=4-tert-butylphenyl) (168 mg, 0.51 mmol) in dichloromethane
(2.0 mL) at room temperature was added diisopropylethylamine (140
mg, 1.07 mmol) followed by methanesulphonyl chloride (122 .mu.L,
1.54 mmol) and the reaction mixture was stirred for 48 hrs. A
saturated solution of sodium hydrogen carbonate (5 mL) was added to
the reaction mixture and the organic phase was removed. The aqueous
phase was extracted with further dichloromethane (5 mL) and the
combined organic phase was washed with water (5 mL) and brine (5
mL), then dried with sodium sulfate, filtered and evaporated to
give structure 13 (R.sup.1=4-tert-butylphenyl, R.sup.2=CH.sub.3)
(200 mg, 100% yield) as a yellow gum. R.sub.f (40-60 petroleum
ether:ethyl acetate (4:1))=0.29; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.27 (s, 9H), 2.22 (s, 3H), 2.98 (s, 3H, SO.sub.2CH.sub.3),
3.91 (s, 3H), 4.92 (s, 2H), 7.22-7.52 (m, 6H, Ar--H), 10.84 (s, 1H,
OH); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 21.1, 31.8, 35.2,
38.3, 49.3, 52.7, 112.3, 121.4, 125.1, 126.8, 128.5, 129.7, 137.3,
151.5, 157.8, 171.4; ESMS m/z 406.1 [M+H].sup.+, 423.2
[M+NH.sub.4.sup.+].sup.+ 811.5 [2M+H].sup.+; LC/MS t.sub.R=10.14
(406.2 [(M+H)].sup.+, 811.3 [(2M+H)].sup.+.
[0102] Synthesis of Structure 14 (R.sup.1=4-tert-butylphenyl,
R.sup.2=CH.sub.3):
[0103] To a stirred solution of structure 13 (200 mg, 0.50 mmol) in
methanol (10 mL) and dichloromethane (10 mL) at room temperature
was added an aqueous solution of sodium hydroxide (1 M, 10 mL) and
stirring was continued for 20 hrs. The reaction mixture was then
neutralized by the drop-wise addition of dilute hydrochloric acid
(1 M). The organic phase was removed and the aqueous phase
extracted with further dichloromethane (15 mL). The combined
organic phase was washed with brine (15 mL) then dried over sodium
sulfate, filtered and evaporated. The crude product was purified on
silica (25 g) with ethyl aceate:40-60 petroleum ether (3:2) then
ethyl aceate:40-60 petroleum ether (3:2) containing acetic acid
(1%), to give structure 14 (R.sup.1=4-tert-butylph- enyl,
R.sup.2=CH.sub.3) (27.2 mg, 14% yield) as a pale yellow solid.
R.sub.f (ethyl acetate: 40-60 petroleum ether (3:2))=0.88; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 1.27 (s, 9H), 2.23 (s, 3H), 2.99
(s, 3H, SO.sub.2CH.sub.3), 4.92 (s, 2H), 7.23, 7.32 (AA'BB', 4H,
8.4, 8.8 Hz), 7.38 (s, 1H, Ar--H), 7.57 (s, 1H, Ar--H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 21.1, 31.9, 35.3, 38.4, 49.4,
125.3, 126.9, 128.5,.129.0, 130.6, 137.3, 138.4, 151.7,158.4,
174.6; ESMS m/z 392.1 [M+H].sup.+, 409.3 [M+NH.sup.+.sub.4].sup.+,
783.4 [2M+H].sup.+.
Example 2
Representative Compounds of Structure (II)
[0104]
2TABLE 2 (II) 15 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
R.sub.6 2-1 --H --H --H --Cl --H --H 2-2 --H --H --CF.sub.3 --H
--CF.sub.3 --H 2-3 --H --H --H --H --Cl --CH.sub.3 2-4 --H --H --H
--Cl --H --CH.sub.3 2-5 --H --H --CH.sub.3 --H --H --Cl 2-6 --H --H
--H --H --H --Cl 2-7 --H --H --F --H --F --H 2-8 --H --H --H
--CH.sub.3 --H --CH.sub.3 2-9 --H --H --H 16 --H --H 2-10 --H --H
--H --(CH.sub.2).sub.7CH.sub.3 --H --H 2-11 --H --H --H
--C(CH.sub.3).sub.3 --H --H 2-12 --H --H --H --H --CF.sub.3 --H
2-13 --H --H --H --H --H --CH.sub.2OH 2-14 --H --H --H --H 17 2-15
--H --H --H 18 --H 2-16 --H --H --H 19 --H --H 2-17 --H --H --H --H
--H 20 2-18 --H --H --H --Br 21 2-19 --H --H --H 22 --H 2-20 --H
--H --H --H --H 23 2-21 --H --H 24 --H --H 25 2-22 --H --H --H --H
--H 26 2-23 --H --H --H --Cl --H 27 2-24 --H --H --H --H --H
--OH
Example 3
Representative Compounds of Structure (III)
[0105]
3TABLE 3 (III) 28 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
R.sub.6 3-1 --CH.sub.3 --H --H --H --H --H
Example 4
Representative Compounds of Structure (IV)
[0106]
4TABLE 4 (IV) 29 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 4-1
--CH.sub.3 --H --H --C(CH.sub.3).sub.3 4-2 --CH.sub.3 --H --H
--C(CH.sub.3).sub.3 Cpd. R.sub.5 R.sub.6 R.sub.7 4-1 --H --H
--CH.sub.3 4-2 --H --H 30
Example 5
Representative Compounds of Structure (V)
[0107]
5TABLE 5 (V) 31 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
R.sub.6 5-1 --H --H --Cl --H --Cl --H 5-2 --H --H --H --H --Cl --Cl
5-3 --H --H --H --H --H --OCF.sub.3 5-4 --H --OCH.sub.3 --H --H --H
--H 5-5 --H --H --H --CH(CH.sub.3).sub.2 --H --H 5-6 --H --H --H 32
--H --H 5-7 --H --H --H 33 --H
Example 6
Representative Compounds of Structure (VI)
[0108]
6TABLE 6 (VI) 34 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
R.sub.6 6-1 --H --H --Cl --H --Cl --H
Example 7
Representative Compounds of Structure (VII)
[0109]
7TABLE 7 (VII) 35 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
R.sub.6 7-1 --H --H --CF.sub.3 --H --CF.sub.3 --H 7-2 --H --H --H
--H --Cl --CH.sub.3 7-3 --H --H --CH.sub.3 --H --H --Cl
Example 8
Representative Compounds of Structure (VIII)
[0110]
8TABLE 8 (VIII) 36 Cpd. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
R.sub.6 8-1 --H --H --H --Cl --H --H
Example 9
Ant Ligand Binding Assay of Representative Compounds
[0111] A competitive binding assay measuring the ability of
compounds in Tables 2-8 to bind to an ANT polypeptide was
performed. A modification of the procedures set forth in U.S. Ser.
No. 09/569,327 entitled "Production of Adenine Nucleotide
Translocator (ANT), Novel ANT Ligands and Screening Assays
Therefor" (incorporated herein by reference) was utilized. In
brief, competitive binding assays were performed using purified
mitochondria from mammalian tissue or from T. ni cells infected
with a baculovirus expressing ANT protein. The mitochondria were
incubated with 0.5 nm of a labeled atractyloside derivative (e.g.,
.sup.1251-ATR) and 20 .mu.M of the compound to be tested or a
control compound. After incubation of the mitochondria preparation
with the compound and the labeled ligand, the reaction is applied
to filter paper, the filter paper washed to remove non-specific
binding, then dried and the bound radioactivity determined via
scintillation counting. Preferably, compounds of the present
invention will displace 50% of the radioactive ligand. To this end,
compounds 2-1, 2-2, 2-4, 2-5, 2-9 through 2-13, 2-18, 2-19, 2-23
and 2-24 in Table 2, compounds 4-1 and 4-2 in Table 4, compounds
5-1, 5-5 and 5-6 in Table 5 and compound 6-1 in Table 6 satisfy
this criteria.
[0112] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0113] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
* * * * *